Project Summary Metastatic castration-resistant prostate cancer (CRPC) is a lethal disease leading to about 30,000 estimated annual deaths in U.S. The majority of CRPCs are driven by androgen receptor (AR) signaling, which represents a key therapeutic target in metastatic CRPC. Despite development of second generation therapeutics targeting the androgen receptor signaling, the resistance to androgen ablation therapies (e.g. enzalutamide and abiraterone) and increased AR transcriptional activity are major drivers of metastatic CRPC, emphasizing a clear need for novel therapies. Recently, we discovered that the Mixed Lineage Leukemia (MLL) protein complex functions as a co-activator of AR and that the interaction of MLL complex with AR is mediated by menin, a scaffold protein required for MLL recruitment to target genes. Knockdown of components of the MLL complex as well as inhibition of the menin-MLL interaction with small molecules we developed abrogates the AR-mediated signaling and inhibits the AR-mediated gene transcription. We also demonstrated that treatment with a small molecule inhibitor targeting the MLL complex effectively and selectively inhibits proliferation of the prostate cancer cells and leads to a suppression of the in vivo tumor growth in the CRPC xenograft models. Based on our findings, we hypothesize that the MLL complex represents an attractive therapeutic target in CRPC and that inhibition of this complex by blocking the menin-MLL interaction may provide a novel therapeutic strategy for advanced prostate cancer. The overall goal of this proposal is to develop very potent small molecule inhibitors targeting the MLL complex with improved potency in prostate cancer models and optimized drug-like properties and to provide a compelling scientific rationale, including detailed mechanistic insight, to facilitate advancing of these compounds as a novel potential treatment for advanced prostate cancer. To achieve this goal, we propose three specific aims: Aim 1: Develop highly potent small molecule inhibitors of the menin-MLL interaction with significantly improved potency in prostate cancer models and optimal in vivo properties. In Aim 2, we propose to study the mechanism of pharmacologic inhibition of the MLL complex in prostate cancer cells, while in Aim 3 we will assess the in vivo efficacy of the menin-MLL inhibitors in mice models of prostate cancer and investigate the mechanism of resistance of response to these compounds in prostate cancer models. Upon successful completion of this project we expect to identify promising candidate compound(s) that could be further developed for clinical use to treat metastatic CRPC.